Synergistic dietary supplement compositions for the prevention, treatment or control of inflammatory disorders

ABSTRACT

A method for treating an inflammation, arthritis, or a joint disease in a patient in need thereof, comprising administering to the patient an herbal composition, where the herbal composition includes a therapeutically effective amount of an extract of Terminalia chebula; a therapeutically effective amount of an extract of Curcuma longa; and a therapeutically effective amount of a non-acidic, water-immiscible organic solvent extract of a Boswellia serrata resin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of parent U.S. application Ser. No.15/229,710 filed on Aug. 5, 2016, which is a continuation-in-part ofparent International Patent Application No. PCT/IN2015/000065, filed onFeb. 2, 2015, now published as WO/2015/118557, which claims priority toIndian Application No. 531/CHE/2014, dated Feb. 5, 2014. The entiredisclosure of each prior application is incorporated by reference hereinin its entirety.

TECHNICAL FIELD OF THE INVENTION

The invention relates to synergistic compositions for the treatment ofInflammation and Arthritis. The composition comprises a therapeuticallyeffective combination of extracts or fractions derived from themedicinal herbs such as Terminalia chebula, Curcuma longa in combinationwith Boswellia serrata non-acidic resin extracts (BNRE) derived fromBoswellia serrata. The synergistic compositions may further containpharmaceutically or nutraceutically acceptable actives, excipients,carriers or diluents. The invention further relates to the methods ofuse of the composition for the treatment of disease conditions relatedto inflammation, which include arthritis, osteoarthritis and joint pain.

BACKGROUND OF THE INVENTION

Terminalia chebula is a gentle purgative, astringent (unripe fruits aremore purgative, ripe ones are more astringent), stomachic, antibilious,alcerative. It is used in prescriptions for treating flatulence,constipation, diarrhea, dysentery, cyst, digestive disorders, vomiting,enlarged liver and spleen, cough and bronchial asthma, and for metabolicharmony. Bark is used as diuretic. The Ayurvedic Pharmacopoeia of India,along with other therapeutic applications, indicated the use of powderof mature fruits in intermittent fevers, chronic fevers, anaemia andpolyuria. The fruits of T. chebula are used in combination with Emblicaofficinalis and T. bellerica (under the name Triphalaa) in the treatmentof liver and kidney dysfunctions. The main purgative ingredient ofTriphalaa is T. chebula. Shikimic, gallic, triacontanoic and palmiticacids, beta-sitosterol, daucosterol, triethyl ester of chebulic acid andethyl ester of gallic acid; a new ellagitannin, terchebulin, along withpunicalagin and teaflavin A have been isolated from the fruits of T.chebula. A new triterpene, chebupentol, and arjungenin, terminoic acidand arjunolic acid were also isolated from the fruit.

The PCT Publication WO2010119294 provides a composition for oraladministration comprising as active ingredient a combination of materialderived from the plants Andrographis paniculata, Tinospora cordifolia,Eclipta alba, Tephrosia purpurea, Vitex negundo, Zinziber officinale,Terminalia chebula and Withania somnifera. Methods for preparing such acomposition and the use of such a composition in therapy of animals arealso provided. The composition is used for treating e.g. arthritis,respiratory infection in animals.

The U.S. Pat. Nos. 7,658,957 and 7,338,674 and PCT Publication No.WO06061675 provide a novel herbal composition for treatment of arthritisand inflammation. The herbal composition comprises a therapeuticallyeffective combination of extracts obtained from the plants Terminaliachebula, Pluchea lanceolata, Desmodium gangeticum, Vitex negunto andZingiber officinale, optionally along with pharmaceutically acceptableadditives. The invention further comprises methods of making the herbalcomposition and methods of use for the treatment of arthritis andinflammation.

The U.S. Publication No. US20080166432 relates to extracts fromTerminalia plant species that are capable of being used in methods formanaging diseases such as cardiovascular disease, diabetes, degenerativeneurological diseases, cancer, age related diseases like amyloidosis,acute pancreatitis, arthritis, atherosclerosis, cancer, heart diseases,inflammatory bowel disease, myocardial infarction, senile dementia,retinal degeneration and senile cataract; owing to the extracts antioxidation potential. The US '432 also relates to extracts fromTerminalia plant species that are capable of being used in methods formanaging various infectious diseases.

Turmeric, the powdered rhizome of the herb Curcuma longa L.(Zingiberaceae), has been used extensively in Indian and Asian cuisineand it is also used as a coloring and flavoring agent. Powderedturmeric, or its extract, is found in numerous commercially availablebotanical supplements. In Ayurvedic medicine, turmeric has traditionallybeen used to treat inflammation, skin wounds and tumors (Ammon and Wahl,1991, Planta Med., 57:1-7). Turmeric extracts have been reported to haveanti-microbial, anti-inflammatory, antioxidant and anticancer effects.In preclinical animal studies turmeric has shown anti-inflammatory,cancer chemopreventive and anti-neoplastic properties (Kelloff et al.,1996, J. Cell. Biochem. Supplement 26:54-71). The best characterized ofthe compounds found in turmeric are curcuminoids, which have been shownto reduce inflammation.

The U.S. Publication No. US20040086581relates to a joint and arthritispain formula and more particularly to a composition to promote healingand relieve arthritis symptoms. The composition comprises atherapeutically effective combination of CMO-Cetyl Myristoleate;MSM-Organic Sulphur; Collagen; Glucosamine; OPC Proanthocyandians;Bromelain Boswellia; Turmeric; Feverfew; Ginger; White willow extract;Manganese Chelate; Sweetener; Maltodextrin; Buffered Vitamin C.

The PCT publication WO2011125070 relates to oral supplementation ofcurcuminoid with essential oil of turmeric to enhance the bioavailability of curcumin for the prophylaxis, treatment, maintenancetherapy and as add-on therapy for disease conditions such as cancer,heart diseases, diabetes, rheumatoid arthritis, osteoarthritis,Alzheimer's disease, inflammatory bowel diseases.

The gum resin of the plant Boswellia serrata (Burseraceae) has long beenin use for the treatment of rheumatoid arthritis and gout by thepractitioners of Ayurvedic medicines in the Indian system of medicine.Various extracts of the gum resin have shown potent anti-inflammatoryand anti-atherogenic activity in laboratory animals [Cuaz-Pérolin etal., Arterioscler Thromb Vasc Biol February 2008]. Incensole acetate, aBoswellia compound isolated was proved to be a NF-kappa B inhibitor anduseful as anti-inflammatory compound [Moussaieff et al., Mol Pharmacol72, 1657-1664, 2007]. The ethanolic extract of the gum resin of B.serrata inhibits the formation of Leukotriene B4 in rat peritonealneutrophils. Leukotriene B4 is one of the important mediators ofinflammatory reactions [Ammon, H. P. T. et al., Planta Medica, 57, 203(1991)]. The extract of Boswellia was found to be a potentanti-arthritic agent [Kimmatkar et al; Phytomedicine. 2003 January;10(1):3-7.], and immune-modulatory agent [Pungle et al; Indian J ExpBiol. 2003 December; 41(12):1460-1462]. The cholesterol lowering actionof Boswellia serrata was also proved [Zutshi U et al, Indian J Pharmac.18, 182-183, 1986]. In fact, a randomized, double blind, placebocontrolled, crossover clinical trial with Boswellia extract on a groupof patients with osteoarthritis of knee exhibited statisticallysignificant mean improvements with respect to reduction in pain,decreased swelling and increased knee flexion [Kimmatkar N, et. al.,Phytomedicine 2003; 10: 3-7]. The efficacy of Boswellia extracts againstchronic colitis [Gupta I, et. al., Planta Med. 2001; 67: 391-395],Crohn's disease [Gerhardt H, et. al., Z Gastroenterol 2001; 39: 11-17]and bronchial asthma [Gupta I, et. al., Eur J Med Res 1998; 3: 511-51]was also reported.

Many different extracts of Boswellia gum resin have been prepared andmost prominent of them are Boswellia serrata acidic extract, Boswelliaserrata non acidic extract and water soluble extract containingpolysaccharides.

The U.S. Pat. No. 5,629,351 relates to a novel fraction comprising amixture of boswellic acids, wherein the fraction exhibitsanti-inflammatory and anti ulcerogenic activities and a process forisolating a boswellic acid fraction and individual boswellic acidstherefrom.

The PCT Patent Publication WO08036932 provides a method for makingcompositions derived from Boswellia species (frankincense or olibanum)having uniquely elevated volatile oil, boswellic acids, andpolysaccharide compounds, particularly, human oral deliveryformulations, and methods for use of such compositions, e.g. fortreating/preventing arthritis, inflammatory disorders, osteoarthritis,rheumatoid diseases and low back pain.

The U.S. Pat. No. 5,888,514 refers to a composition for treating amammal having a condition characterized by bone or joint inflammationwhere extract of Boswellia serrata is used as one of the ingredients.

The U.S. Publication No. US2012301432 provides Boswellia low polar gumresin extract (BLPRE) comprising a novel phytochemical composition aloneand its compositions for the prevention, control and treatment ofdisorders such as diabetes, obesity, metabolic syndrome, excess bodyweight, inflammation, asthma, Alzheimer's, cognitive disorders,neurological disorders, cartilage degradation, aging, skin disorders,hypertriglyceridemia, hyperlipidemia, hypercholesterolemia, cholesteroldisorders, hypertension, high blood pressure, immune disorders, cancer,coronary heart disease, infectious diseases, osteoporosis,osteoarthritis, rheumatoid arthritis, joint pain, joint discomfort andseveral other conditions associated thereof.

The PCT Publication No. WO201029578 discloses synergistic nutraceuticalanti-inflammatory compositions comprising therapeutically effectivecombination of an extract selectively enriched in3-O-acetyl-11-keto-β-boswellic acid (AKBA) derived from Boswelliaserrata and Boswellia serrata non-acidic resin extract (BNRE). Thecompositions can be used to prevent, control and treat inflammation andseveral inflammatory related diseases including asthma, osteoarthritis,rheumatoid arthritis, endothelial dysfunction and the like. Theinvention further discloses the amelioration of pro-inflammatorybiomarker proteins or molecules, whose expression/production is alteredduring inflammatory diseases.

Inflammation is a complex pathophysiological process mediated by avariety of signaling molecules produced by leucocytes, macrophages andmast cells as well as by the activation of complement factors that bringabout edema formation as a result of extravasations of fluid andproteins and accumulation of leucocytes at the inflammatory site.Arthritis is a disease affecting the musculoskeletal system,particularly the joints. Inflammation is a common clinical conditionsand rheumatoid arthritis (RA) is a chronic debilitating autoimmunedisorder that affects about 3 million Indians and 50 million Americansetc. Osteoarthritis is the most common joint degenerative disorder setsin as a result of aging, wear and tear on a joint.

Accordingly, there is a real and continuing need in the art for safe andcost-effective medicines for treating inflammation and arthritis. Hencethe aim of present invention is to develop synergistic compositions,specifically those containing the herbal extracts and/or bioactivefractions to treat the Inflammation and Arthritis without the undesiredside effects. In particular, the invention provides compositionscomprising extracts or fractions derived from Terminalia chebula,Curcuma longa and Boswellia serrata.

There is however no prior art, to the best of inventors' knowledge,relating to the compositions comprising extracts derived from Terminaliachebula and Curcuma longa in combination with Boswellia serratanon-acidic resin extracts (BNRE) for the prevention, control andtreatment of inflammatory conditions and joint disorders.

Therefore, the main object of the present invention is to providesynergistic nutraceutical or dietary supplement compositions comprisingtherapeutically effective combination of extracts or fractions fromTerminalia chebula, Curcuma longa and Boswellia serrata non-acidic resinextract (BNRE), optionally containing other natural ingredients.

SUMMARY OF THE INVENTION

In accordance with the above object, the invention discloses synergisticcompositions for the prevention, control or treatment of inflammationand/or disease conditions associated with or related to inflammationincluding asthma, atherosclerosis, endothelial dysfunction,osteoarthritis, rheumatoid arthritis and the like.

In another aspect, the invention provides synergistic compositionscomprising therapeutically effective combination of extracts orfractions from Terminalia chebula, Curcuma longa and Boswellia serratanon-acidic resin extract (BNRE) for the prevention, control andtreatment of one or more components of other inflammatory diseasesincluding arthritis or rheumatoid arthritis. Non-limiting examples ofarthritis include rheumatoid (such as soft-tissue rheumatism andnon-articular rheumatism, fibromyalgia, fibrositis, muscular rheumatism,myofascial pain, humeral epicondylitis, frozen shoulder, Tietze'ssyndrome, fascitis, tendinitis, tenosynovitis, bursitis), juvenilechronic, joint disorders, spondyloarthropathies (ankylosingspondylitis), osteoarthritis, hyperuricemia and arthritis associatedwith acute gout, chronic gout and systemic lupus erythematosus anddegenerative arthritis.

In yet further aspect, the present invention provides synergisticcompositions for the amelioration of the expression and/or production ofbiomolecules or biomarkers associated with inflammation and/or diseaseconditions associated with inflammation, osteoarthritis, rheumatoidarthritis, cartilage degradation, which include but not limited to5-lipoxygenase (5-LOX), 5-Lipoxygenase activating protein (FLAP),macrophage/adipocyte fatty acid-binding protein-2 (aP2/FABP),interferon-gamma (IFN-γ), interleukin-4 (IL-4), intercellular celladhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1,Matrix metalloproteinase (MMP)-3, TNF-α, and IL-1β in mammals.

Various embodiments disclosed herein relate to a herbal composition,comprising:

a therapeutically effective amount of an extract of Terminalia chebula;

a therapeutically effective amount of an extract of Curcuma longa; and

a therapeutically effective amount of a non-acidic, water-immiscibleorganic solvent extract of a Boswellia serrata resin.

In various embodiments, the non-acidic, water-immiscible organic solventextract of the Boswellia serrata resin is obtained by:

extracting Boswellia serrata gum resin with a water-immiscible organicsolvent to obtain an organic solution;

extracting the organic solution with aqueous alkali to remove boswellicacids from the organic solution; and

removing the water-immiscible organic solvent from the organic solutionto obtain an organic solvent extract. In some embodiments, the method ofobtaining the non-acidic, water-immiscible organic solvent extract ofthe Boswellia serrata resin further comprises a step of removingvolatile components from the organic solvent extract under vacuum toobtain the desired non-acidic, water-immiscible organic solvent extract.

In some embodiments, the composition further comprises a plant derivedanti-inflammatory agent, a pharmaceutically, nutraceutically, ordietetically acceptable carriers, a pharmaceutically, nutraceutically,or dietetically acceptable excipient, or a mixture thereof.

In some embodiments, the composition comprises an extract of Terminaliachebula prepared from dried fruits of Terminalia chebula; an extract ofCurcuma longa prepared from dried rhizomes of Curcuma longa; and anon-acidic, water-immiscible organic solvent extract of a Boswelliaserrata resin prepared from the gum resin of Boswellia serrata.

According to various embodiments disclosed herein, the compositioncomprises from 20% by weight to 75% by weight of the extract ofTerminalia chebula. The composition may comprise from 10% by weight to40% by weight, or from 15% by weight to 40% by weight, of the extract ofCurcuma longa. The composition may comprise from 10% by weight to 40% byweight of the non-acidic, water-immiscible organic solvent extract ofthe Boswellia serrata resin. The composition may further comprise from0% by weight to 20% by weight of a Boswellia serrata extract whichcontains at least 5% 3-O-acetyl-11-keto-β-boswellic acid (AKBA). Theabove percentages are based on the total weight of the Terminaliachebula, Curcuma longa, and Boswellia serrata extracts.

In some embodiments, the composition comprises from 25% by weight to 65%by weight, or from 25% by weight to 45% by weight, of the extract ofTerminalia chebula. The composition may comprise from 12% by weight to30% by weight, or from 15% by weight to 25% by weight, of the extract ofCurcuma longa. The composition may comprise from 12% by weight to 30% byweight, or from 15% by weight to 25% by weight, of the non-acidic,water-immiscible organic solvent extract of the Boswellia serrata resin.The composition may further comprise from 15% by weight to 25% by weightof a Boswellia serrata extract which contains at least 5%3-O-acetyl-11-keto-β-boswellic acid (AKBA). The above percentages arebased on the total weight of the composition.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail in connection with certainpreferred and optional embodiments, so that various aspects thereof maybe more fully understood and appreciated.

Source of the Plant Material

The herbs Terminalia chebula, Curcuma longa and Boswellia serrata usedin the present invention are collected from Eastern India, AndhraPradesh and Madhya Pradesh respectively.

Inflammation is a response of the vascular tissues to stimuli such aspathogens, damaged cells or allergic agents, which enter into the body.It is a protective mechanism by the organism to remove harmful pathogensor agents and protect the tissues. Pro-inflammatory cytokines such asTNFα, IL-1β, IL-6, GM-CSF and CD4+, Th2 subset derived IL-4, IL-5 andIL-13 lymphokines are considered as the key factors of immunepathogenesis of inflammatory diseases [Knight D A, et. al., J. AllergyClin. Immunol. 2001; 108: 797-803]. 5-Lipoxygenase is an enzyme criticalfor leukotriene synthesis from arachidonic acid, a key step in theinflammatory process. Leukotrienes are key mediators of inflammatorydiseases.

The inventors have prepared several plant extracts and tested them foranti-oxidant activity using Reactive Oxygen Species (ROS) scavengingassay; and anti-inflammatory activities (5-Lipoxygennase inhibition andinhibition of cytokines in cellular models) for identifying activeextracts. Several plant extracts including Terminalia chebula fruitextract, Curcuma longa root extract, Boswellia serrata non-acidic gumresin extract and Boswellia serrata gum acidic resin extract showedpromising activity during the screening. The inventors have randomlyprepared and tested several compositions comprising the extracts ofTerminalia chebula fruit in combination with several plant extracts inan effort to find a composition that can show better efficacy inameliorating the inflammatory processes and in expression or productionof key biomarker molecules associated with inflammatory disorders, andto identify a better agent for controlling, treating and preventinginflammatory disease conditions. Several enzyme based and cell basedin-vitro anti-inflammatory studies were conducted on a broad array ofcompositions containing Terminalia chebula in combination with severalplant extracts. It was found surprisingly found that compositionscomprising extract(s) or fractions or enriched fraction(s) orcompound(s) derived from Terminalia chebula in combination with extractsor enriched fractions or compounds derived from Curcuma longa andBoswellia serrata non-acidic resin extract (BNRE) potently inhibited the5-lipoxygenase enzyme (5-LOX) activity. The water extract of Terminaliachebula fruit, methanol extract of Curcuma longa and Boswellia serratanon-acidic resin extract (BNRE) a non-acidic gum resin extract obtainedafter removing/separating the acidic compounds and volatile compoundsfrom the water immiscible organic solvent extract of Boswellia serratagum resin, such as Methyl isobutyl ketone were used to demonstrate theinvention. However, extracts obtained using other solvents such asdichloromethane, dichloroethane, ethanol, methanol; water and mixturesthereof; ethyl acetate; C1 to C7 hydrocarbons; hydroalchohol andmixtures can also be used to inhibit the 5-lipoxygenase enzyme (5-LOX)activity.

The individual extracts and the compositions were tested for theirefficacy to inhibit 5-lipoxygenase enzyme (5-LOX) activity in an enzymebased assay. The individual extracts and compositions were also testedfor their efficacy to inhibit productions of a few key pro-inflammatorycytokines/chemokines such as IL-1β, TNF-α and MMP-3 in in vitro cellularmodels. It was found very surprisingly that compositions comprisingtherapeutically effective combination of extracts or fractions fromTerminalia chebula and Curcuma longa, and Boswellia serrata non-acidicresin extract (BNRE) showed synergistic inhibition of 5-LOX activity.

The individual extracts and the compositions comprising therapeuticallyeffective combination of extracts or fractions from Terminalia chebulaand Curcuma longa, Boswellia serrata non-acidic resin extract (BNRE) andBoswellia serrata extract standardized to 75%-85% total acids byvolumetric assay which contain at least 5%3-O-acetyl-11-keto-β-boswellic acid (AKBA) were tested for theirefficacy to inhibit 5-lipoxygenase enzyme (5-LOX) activity, andproduction of key pro-inflammatory cytokines/chemokines such as IL-1β,TNF-α and MMP-3. It was found very surprisingly that compositionscomprising therapeutically effective combination of extracts orfractions from Terminalia chebula and Curcuma longa, Boswellia serratanon-acidic resin extract (BNRE) and Boswellia serrata extractstandardized to 75%-85% total acids by volumetric assay which contain atleast 5% 3-O-acetyl-11-keto-β-boswellic acid (AKBA) showed synergisticinhibition of 5-LOX activity and the cytokines/chemokines production.

In a preferred embodiment, the present invention provides synergisticanti-inflammatory compositions comprising therapeutically effectivecombination of extracts or fractions derived from Terminalia chebula andCurcuma longa, and non-acidic resin extract (BNRE) derived fromBoswellia serrata.

In other important embodiment, the compositions comprisingtherapeutically effective combination of extracts or fractions derivedfrom Terminalia chebula and Curcuma longa, and non-acidic resin extract(BNRE) derived from Boswellia serrata can be used to prevent or cure ortreat inflammation and/or one or more disease conditions related to orassociated with inflammation.

In another aspect, the present invention provides synergisticanti-inflammatory compositions obtained by combining Terminalia chebulaand Curcuma longa derived extracts or fractions and Boswellia serratanon-acidic resin extract (BNRE) at certain specific ratio to obtain abetter effect than that obtained when the extracts or fractions weregiven individually, i.e. synergistic amelioration of inflammatoryprocesses and/or expression or production of pro-inflammatorybiomolecules/protein markers.

In another preferred embodiment, the synergistic anti-inflammatorycompositions comprising therapeutically effective combination ofextracts or fractions derived from Terminalia chebula and Curcuma longa,and Boswellia serrata non-acidic resin extract (BNRE) optionally containBoswellia serrata acidic extract standardized to one or more boswellicacid compounds.

In yet another preferred embodiment, the present invention provides asynergistic composition consisting of Terminalia chebula extract orfraction in the range from 20% to 75 by weight, Curcuma longa extract orfraction in the range from 10% to 40% by weight and Boswellia serratanon-acidic resin extracts (BNRE) in the range from 10% to 40% by weight.

In a further aspect, the invention provides synergistic compositionscontaining extracts or fractions derived from Terminalia chebula andCurcuma longa and Boswellia serrata non-acidic resin extract (BNRE) forthe amelioration of the expression or production of thebiomolecules/biomarkers/certain redox-sensitive pro-inflammatory genesrelated to or associated with inflammation which include but not limitedto 5-lipoxygenase (5-LOX), 5-Lipoxygenase activating protein (FLAP),macrophage/adipocyte fatty acid-binding protein (aP2/FABP), IFN-γ, IL-4,ICAM-1, VCAM-1, Matrix metalloproteinases (MMPs) such MMP-13, MMP-3 andMMP-1, NFxB TNF-α, and IL-1β, IL-13, IL-6, IL-2,in mammals in needthereof.

In yet another aspect, the invention provides synergistic compositionscontaining extracts or fractions derived from Terminalia chebula andCurcuma longa and Boswellia serrata non-acidic resin extract (BNRE) forthe prevention, control and treatment of one or more disease conditionsrelated to or associated with inflammation which include but not limitedto asthma, arthritis, osteoarthritis, rheumatoid arthritis,atherosclerosis, endothelial dysfunction, allergic rhinitis, dermatitis,psoriasis, cystic fibrosis, inflammatory bowel diseases, interstitialcystitis, migraines, angina, chronic prostatitis, sun burn, periodontaldisease, multiple sclerosis, uveitis, post-angioplasty restenosis,glomerulonephritis, gastrointestinal allergies, nephritis,conjunctivitis, chronic obstructive pulmonary disease, occupationalasthma, eczema, bronchitis, hay fever, hives, allergic disorders and forconditions like wheezing, dyspnea, non-productive cough, chesttightness, neck muscle tightness, rapid heart rate, chest pain, jointpain, joint disorders, collagen degradation by UV irradiation,skin-wrinkling and skin-aging and several other conditions associatedthereof in mammals.

Non-limiting examples of arthritis include rheumatoid (such assoft-tissue rheumatism and non-articular rheumatism, fibromyalgia,fibrositis, muscular rheumatism, myofascial pain, humeral epicondylitis,frozen shoulder, Tietze's syndrome, fascitis, tendinitis, tenosynovitis,bursitis), juvenile chronic, joint disorders, spondyloarthropathies(ankylosing spondylitis), osteoarthritis, hyperuricemia and arthritisassociated with acute gout, chronic gout and systemic lupuserythematosus and degenerative arthritis.

In a further embodiment, the present invention provides the process forproducing extracts or fractions and their compositions from Terminaliachebula, Curcuma longa and Boswellia serrata non-acidic resin extract(BNRE) plant parts including but not limited to bark, root, stem,leaves, fruit, gum resin, rhizome and mixtures thereof.

In another embodiment, the solvents that can be used for preparing theextracts or fractions of the herbs can be selected from chlorinatedsolvents, such as, dichloromethane and dichloroethane, C1-C5 alcoholssuch as ethanol, methanol; water and mixtures thereof; esters such asethyl acetate; C1 to C7 hydrocarbons; hydroalchohol and mixturesthereof.

In yet another embodiment of the present invention, the synergisticcompositions containing extracts or fractions derived from Terminaliachebula and Curcuma longa and Boswellia serrata non-acidic resin extract(BNRE) contain optionally one or more of pharmaceutically ornutraceutically or dietetically acceptable excipient(s) or diluents orsalt(s) or additive(s).

In yet another embodiment of the present invention, the synergisticcompositions derived from Terminalia chebula and Curcuma longa andBoswellia serrata non-acidic resin extract (BNRE) are produced fromaqueous, alcoholic or hydro alcoholic extracts.

In yet another embodiment of the present invention, the synergisticcompositions derived from Terminalia chebula and Curcuma longa andBoswellia serrata non-acidic resin extract (BNRE) and Boswellia serrataextract standardized to 75%-85% total acids by volumetric assay whichcontain at least 5% 3-O-acetyl-11-keto-β-boswellic acid (AKBA).

In yet another embodiment of the present invention, the synergisticcompositions comprises25-45% of Terminalia chebula, 15-25% of Curcumalonga and 15-25% of Boswellia serrata non-acidic resin extract (BNRE)and 15-25% of Boswellia serrata extract standardized to 75%-85% totalacids by volumetric assay which contain at least 5%3-O-acetyl-11-keto-β-boswellic acid (AKBA) and 5-15% of excipients

In yet another embodiment of the present invention, the synergisticcompositions composition comprises 36% of Terminalia chebula extract,18% of Curcuma longa extract, 18% Boswellia serrata non-acidic resinextract (BNRE) and 18% of Boswellia serrata extract standardized to75%-85% total acids by volumetric assay which contain at least 5%3-O-acetyl-11-keto-β-boswellic acid (AKBA) and 10% of excipients.

The other embodiments of the present invention further provides theusage of the said synergistic compositions as it is or in comminutedform and/or in unmodified form as granules or powder or paste or theactive ingredients are formulated into a solid, semi-solid or liquiddosage form by adding a conventional biologically or pharmaceuticallyacceptable salt(s) or additive(s) or excipient(s).

In a further embodiment, the invention provides therapeuticallyeffective amount of the novel compositions of the present inventionwhich can be administered in a specific dosage form such as orally,topically, transdermally, parenterally or in the form of a kit to asubject or patient in need thereof. Specific dosage form for formulationof the compositions of the present invention includes but not limited tooral agents such as tablets, soft capsules, hard capsules, pills,granules, powders, infusion solution, injection solution, cream, gel,emulsions, ointment, enema, medicinal pack, food supplement, emulsions,suspensions, syrups, pellets, inhalers, mouth sprays and the like; andparenteral agents such as injections, drops, suppositories and the like.

In other preferred embodiment, the present invention provides a methodfor the prevention, control and treatment of one or more diseaseconditions related to or associated with inflammation which include butnot limited to asthma, arthritis inflammatory bowel disease, rheumatoidarthritis, juvenile rheumatoid arthritis, psoriatic arthritis,osteoarthritis, refractory rheumatoid arthritis, chronic non-rheumatoidarthritis, atherosclerosis, endothelial dysfunction, allergic rhinitis,dermatitis, psoriasis, cystic fibrosis, inflammatory bowel diseases,interstitial cystitis, migraines, angina, chronic prostatitis, sun burn,periodontal disease, multiple sclerosis, uveitis, post-angioplastyrestenosis, glomerulonephritis, gastrointestinal allergies, nephritis,conjunctivitis, chronic obstructive pulmonary disease, occupationalasthma, eczema, bronchitis, hay fever, hives, allergic disorders and forconditions like wheezing, dyspnea, non-productive cough, chesttightness, neck muscle tightness, rapid heart rate, chest pain, jointpain, collagen degradation by UV irradiation, skin-wrinkling andskin-aging and several other conditions associated thereof in mammals,wherein the method comprises supplementing said mammal with an effectiveamount of synergistic composition containing extracts or fractionsderived from Terminalia chebula fruit and Curcuma longa root andnon-acidic resin extract derived Boswellia serrata.

In a further embodiment of the present invention, the compositions mayfurther comprises an effective amount(s) of one or more pharmaceuticalor nutraceutical or dietetically acceptable agents including but notlimited to antioxidant(s), adaptogen(s), anti-inflammatory agent(s),anti-diabetic agent(s), antiobese agent(s), anti-atheroscleroticagent(s), bio-protectants and/or bio-availability enhancer(s) and tracemetals or an excipient(s) or pharmaceutically acceptable salt(s) oradditive(s) and the compositions or mixtures thereof to form aformulation(s) that can be administered using any of the methodsdescribed above.

The examples of pharmaceutically acceptable anti-inflammatory agentsemployed in the present invention include, but are not limited toprednisolone, hydrocortisone, methotrexate, sulfasalazine, naproxen,diclofenac and ibuprofen.

The examples of the biologically or pharmaceutically acceptable carriersemployed in the present invention include, but are not limited to,surfactants, excipients, binders, diluents, disintegrants, lubricants,preservatives, stabilizers, buffers, suspensions and drug deliverysystems.

Preferred examples of solid carriers or diluents or excipients includebut not limited to glucose, fructose, sucrose, maltose, yellow dextrin,white dextrin, aerosol, microcrystalline cellulose, syloid, calciumstearate, magnesium stearate, sorbitol, stevioside, corn syrup, lactose,citric acid, tartaric acid, malic acid, succinic acid, lactic acid,L-ascorbic acid, dl-alpha-tocopherol, glycerin, propylene glycol,glycerin fatty ester, poly glycerin fatty ester, sucrose fatty ester,sorbitan fatty ester, propylene glycol fatty ester, acacia, carrageenan,casein, gelatin, pectin, agar, vitamin B group, nicotinamide, calciumpantothenate, amino acids, calcium salts, pigments, flavors andpreservatives.

Preferred examples of liquid carriers (diluents) include, distilledwater, saline, aqueous glucose solution, alcohol (e.g. ethanol),propylene glycol and polyethylene glycol; and oily carriers such asvarious animal and vegetable oils, white soft paraffin, paraffin andwax.

In alternative aspect of the invention, the inventive compositions ofthe present invention are delivered in the form of controlled releasetablets, using controlled release polymer-based coatings by thetechniques including nanotechnology, microencapsulation, colloidalcarrier systems and other drug delivery systems known in the art. Thesaid compositions can be designed for once a daily administration ormultiple administrations per day.

In accordance to the present invention, the compositions of the presentinvention can also be formulated into or added to existing or new foodand beverage form(s) such as solid foods like cereals, baby food(s),chocolate or nutritional bars, semisolid food like cream or jam, or gel,refreshing beverage, coffee, tea, milk-contained beverage, dairyproducts, lactic acid bacteria beverage, soup, drop, candy, chewing gum,chocolate, gummy candy, yoghurt, ice cream, pudding, soft adzuki-beanjelly, jelly, cookie, bakery products and the like. These variouscompositions or preparations or foods and drinks are useful as a healthyfood for the treatment and/or prevention of inflammation and/or one ormore of disease conditions associated with or related to inflammationincluding but not limited to asthma, arthritis inflammatory boweldisease, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriaticarthritis, osteoarthritis, refractory rheumatoid arthritis, chronicnon-rheumatoid arthritis, osteoporosis/bone resorption, coronary heartdisease, atherosclerosis, vasculitis, ulcerative colitis, psoriasis,adult respiratory distress syndrome, diabetes, metabolic disorders,delayed-type hypersensitivity in skin disorders and Alzheimer's disease.

Various exemplary embodiments of the invention provides that the amountof present synergistic compositions to be administered or supplementedto humans or mammals may not be uniform and varies depending on thenature of the formulation and suggested human or animal dosage of theextract or the fractions, but preferably, within a range from 0.1 to 750mg/kg body weight per day, more preferably about 0.5 to 500 mg/kg bodyweight.

Another embodiment of the invention provides that the quantity of thepresent inventive compositions in the above-mentioned variousformulations, food and beverage compositions may also not be uniform andvaries depending on the nature of the formulation and suggested human oranimal dosage of the compositions, for example, about 0.001% to 99%,more preferably about 0.1 to 90 wt %.

In one of the preferred embodiments, the concentration of Terminaliachebula extract varies in the range of 20% to 75% by weight.

In another preferred embodiment, the concentration of Curcuma longaextract varies in the range of 10% to 40% by weight. In anotherpreferred embodiment, the concentration of Curcuma longa extract variesin the range of 15% to 40% by weight.

In yet another preferred embodiment, the concentration of Boswelliaserrata non-acidic resin extract (BNRE) varies in the range of 10% to40% by weight.

In still another embodiment, the synergistic composition comprisesTerminalia chebula by weight ranging from 20% to 75%, Curcuma longa byweight ranging from 10% to 40% and Boswellia serrata non-acidic resinextracts (BNRE) by weight ranging from 10% to 40%.

In some embodiments, the synergistic composition comprises Terminaliachebula by weight ranging from 20% to 75%, Curcuma longa by weightranging from 15% to 40% and Boswellia serrata non-acidic resin extracts(BNRE) by weight ranging from 10% to 40%.

In yet another embodiment, the synergistic composition comprisesTerminalia chebula by weight ranging from 25% to 65%, Curcuma longa byweight ranging from 12% to 30% and Boswellia serrata non-acidic resinextracts (BNRE) by weight ranging from 12% to 30%.

Further embodiment of the invention provides that the amount of thepresent synergistic composition(s) varies in the range of 1% to 100% byweight based on the total weight of the composition.

In another embodiment, the invention further comprises; mixing thecompositions of the present invention with various components used inthe animal feed for the purpose of curing, preventing or treatinginflammation and several inflammation associated or related diseasesincluding asthma, atherosclerosis and arthritis and the like.

The form of the composition or formulation to be added to animal feed isnot specifically limited and may be added it is, or as a composition(s),to various cooked and processed food products. The quantity may be thesame as that used in case of food products. Similarly, the ingredientsmay also be added during or after preparation of the animal feeds.

In accordance to the present invention, the compositions of the presentinvention can also be formulated into any dietary supplement, food andbeverage forms for human and animal applications.

In another preferred embodiment, the invention provides a method fortreating an inflammation, arthritis and joint disease in a patient inneed thereof comprising administering to the patient an effective amountof novel synergistic composition(s) comprising a combinations ofTerminalia chebula extract, Curcuma longa extract and Boswellia serratanon-acidic resin extract (BNRE) optionally comprises at least oneingredient selected from plant derived anti-inflammatory agents,pharmaceutically or nutraceutical or dietetically acceptablecarriers/excipients.

The method for treating an inflammation, arthritis and joint disease areselected from rheumatoid arthritis, psoriatic arthritis, osteoarthritis,refractory rheumatoid arthritis, chronic non-rheumatoid arthritis,osteoporosis/bone resorption, collagen degradation, gouty arthritis,lupus or juvenile arthritis and Joint pain.

The method of treating an inflammatory condition which include but notlimited to rheumatoid arthritis, psoriatic arthritis, osteoarthritis,refractory rheumatoid arthritis, chronic non-rheumatoid arthritis,osteoporosis/bone resorption, collagen degradation, gouty arthritis,lupus or juvenile arthritis and Joint pain in a mammal, where in themethod comprises, supplementing the mammal in need there off asynergistic compositions comprising 25-45% of Terminalia chebula, 15-25%of Curcuma longa and 15-25% of Boswellia serrata non-acidic resinextract (BNRE) and 15-25% of Boswellia serrata extract standardized to75%-85% total acids by volumetric assay which contain at least 5%3-O-acetyl-11-keto-β-boswellic acid (AKBA) and 5-15% of excipients.

In another preferred embodiment, the invention provides use of novelsynergistic composition(s) comprising a combinations of Terminaliachebula extract, Curcuma longa extract and Boswellia serrata non-acidicresin extract (BNRE) optionally comprises at least one ingredientselected from plant derived anti-inflammatory agents, pharmaceuticallyor nutraceutical or dietetically acceptable carriers/excipients, fortreating an inflammation, arthritis and joint disease.

The following examples, which include preferred embodiments, will serveto illustrate the practice of this invention, and it being understoodthat the particulars shown are by way of example and for purpose ofillustrative discussion of preferred embodiments of the invention andthey are not to limit the scope of the invention.

Example 1: Preparation of Terminalia chebula Water Extract

Dried fruits of plant Terminalia chebula (1 Kg) were pulverized tocoarse powder, extracted with water (5L) at 80° C. for 1.5 hour. Theextraction process was repeated thrice using water (5, 3.5, and 3.5 L)in 1:3-1:5 ratio w/v with respect to plant material. All the extractswere combined, the combined aqueous extracts were fine filtered, and theclear extract was evaporated to dryness on a climbing film evaporator at50-60° C. to obtain 300 g of Terminalia chebula extract.

Example 2: Preparation of Terminalia chebula Alcohol Extract

Dried fruits of plant Terminalia chebula (0.1 Kg) were pulverized tocoarse powder, extracted with alcohol for 1 hrs at Rt/at reflux temp.Extraction process repeated thrice using with Alcohol are in the ratio1:12-1:17 W/V with respect to the plant material. All the extracts werecombined, the combined alcohol extracts were fine filtered, and theclear extract was evaporated under at 40° C. under vacuum to give drypowder (290 gm).

Example 3: Preparation of Curcuma longa Alcohol Extract

Dried rhizomes of plant Curcuma longa (1 Kg) was pulverized to coarsepowder, extracted with 5 L of alcohol such as ethanol, methanol ormixtures thereof at temperature range 60-65° C. for 2-3 hrs. Extractionprocess repeated thrice using alcohol in the ratio 1:4-1:5 w/v withrespect to the plant material. All the three extractions were combinedand fine filtered. The filtrate was evaporated to dryness under reducedpressure at 40-50° C. to obtain 70 g of Curcuma longa extract whenmethanol is the extraction medium.

Example 4: Preparation of Boswellia serrata Non-Acidic Resin Extract(BNRE)

The Boswellia serrata gum resin (100 g) was dispersed in 600 mL ofmethyl isobutyl ketone (MIBK) solvent and stirred at room temperaturefor 6.0 min. The insoluble gum materials were separated by filtration.The MIBK solution was extracted repeatedly with 2% KOH solution (3×200mL) to remove the acidic compounds. The MIBK layer was then washedsuccessively with water (400 mL) and brine (200 mL). The MIBK layer wasevaporated under reduced pressure at 60-70° C. and the volatilecomponents are removed from the oily residue under vacuum at 120-150° C.to obtain Boswellia serrata non-acidic resin extract or BNRE as aviscous oil (12 g).

The aqueous alkali solution containing the acidic compounds wasacidified using concentrated HCl solution. The precipitate obtained wasfiltered and solid dried under vacuum to obtain Boswellia serrata acidicextract, which showed 75%-85% total acids by volumetric assay.

The above procedure can also be performed using ethyl acetate in placeof MIBK to obtain non-acidic resin extract and acidic resin extracts.

Alternatively, the gum resin (250 g) collected from Boswellia serratawas extracted with methanol (300 mL×3) and the combined methanol extractwas concentrated. The residue (50 g) was dissolved in ethyl acetate (400mL) and extracted thrice with 2N KOH (3×100 mL). The organic layer waswashed with water (2×200 mL) and brine (200 mL) and evaporated to obtainBoswellia oil. The volatile compounds were evaporated from the oil undervacuum at 120-150° C. to obtain 22 g of Boswellia serrata non-acidicresin extract (BNRE).

Example 5: Preparation of Boswellia serrata Acidic Extract

Raw material 0.1 kg was extracted with 800 mL of Ethyl Acetate (EtOAc)under reflux for 3 hrs. Extraction process repeated with two times usingEtOAc in the 1:6 w/v ratios with respect to plant material. EtOAcsoluble extractives were treated with 2.8% Aq. KOH (3×1:1 v/v withrespect with EtOAc extract). The mixture was stirred for 30 min andsettled for 2 hrs. Separate the top EtOAc layer and washed successivelywith water and brine 1:4 v/v and 1:2 v/v with respect to EtOAc extractrespectively. Top layer (Ethyl acetate) was concentrated at 70-75° C.for 3-4 hrs and further concentrated at 140° C. under high vacuum for 2hrs (HPR5:1).

Bottom layer (base layer) was concentrated at 70-75° C. under vacuum for3 hrs. and diluted with water. Adjusted pH to 1.45 with 10% HCl and theprecipitate filtered and washed with water till neutral pH. Wet cakedried under vacuum for 20 hrs to obtain acidic gum resin extract (130gm).

Example 6: Composition-1

The composition-1 was prepared by combining the water extract ofTerminalia chebula, methanol extract of Curcuma longa and Boswelliaserrata non-acidic resin extract (BNRE) in the ratio of 2:1:1(50%:25%:25%).

Example 7: Composition-2

The composition-2 was prepared by combining the water extract ofTerminalia chebula, methanol extract of Curcuma longa and Boswelliaserrata non-acidic resin extract (BNRE) in the ratio of 7.5: 1.5: 1.0(75%:15%:10%).

Example 8: Composition-3

The composition-3 was prepared by combining the water extract ofTerminalia chebula, methanol extract of Curcuma longa and Boswelliaserrata non-acidic resin extract (BNRE) in the ratio of 7.5:1.0:1.5(75%:10%:15%).

Example 9: Composition-4

The composition-4 was prepared by combining the water extract ofTerminalia chebula, methanol extract of Curcuma longa and Boswelliaserrata non-acidic resin extract (BNRE) in the ratio of 1:2:2(20%:40%:40%).

Example 10: Composition-5

The composition-5 was prepared by combining the water extract ofTerminalia chebula, methanol extract of Curcuma longa, Boswellia serratanon-acidic resin extract (BNRE) and Boswellia serrata extractstandardized to 75%-85% total acids by volumetric assay which contain 5%3-O-acetyl-11-keto-β-boswellic acid (AKBA) in the ratio of 2:1:1:1(40%:20%:20%:20%).

Example 11: Composition-6

The composition-6 was prepared by combining the water extract ofTerminalia chebula, methanol extract of Curcuma longa, Boswellia serratanon-acidic resin extract (BNRE), and excipients [Microcrystallinecellulose (MCC) and Syloid] in the ratio of 2:1:1:2 (34%:17%:17%:32%).

Example 12: Composition-7

The composition-7 was prepared by combining the water extract of 36 g ofTerminalia chebula, 18 g of methanol extract of Curcuma longa, 18 g ofBoswellia serrata non-acidic resin extract (BNRE), 18 g of Boswelliaserrata extract standardized to 75%-85% total acids by volumetric assaywhich contain 5% 3-O-acetyl-11-keto-β-boswellic acid (AKBA) and 10 g ofexcipients [Microcrystalline cellulose (MCC) and Syloid], so that theirpercentages in the composition are 36%, 18%, 18%, 18% and 10%respectively.

Example 13: 5-Lipoxygenase Enzyme (5-LOX) Inhibitory Activity

5-Lipoxygenase enzyme (5-LOX) inhibitory activity was measured using themethod of Lip Yong Chung et al., (Pharmaceutical Biology, Vol. 47 (12),1142-1148, 2009). The assay mixture contained 80 μM linoleic acid andsufficient amount of potato 5-lipoxygenase in 50 mM TrisHCl buffer (pH7.4). 5 μL of 5-LOX enzyme was added to 175 μL of 50 mM TrisHCl buffer.The reaction was initiated by the addition of 5 μL linoleic acid (finalconc. 140 μM) in 50 mM TrisHCl buffer followed by incubation at 250 C indark 20 min. The total volume of the reaction mixture is 185 μL. Theassay was terminated by the addition of 65 μL freshly prepared foxreagent. After incubation for 20 min the absorbance was read using XmarkMicro plate spectrophotometer (BIO-RAD) at 595 nm. The reaction wasmonitored for 120 sec and the inhibitory potential of the testsubstances i.e., extracts and compositions was measured by incubatingvarious concentrations of test substances two minutes before theaddition of linoleic acid. All assays were performed three times.Percentage inhibition of 5-Lipoxygenase activity was calculated bycomparing slope of the curve obtained for test substances with that ofthe control. The half maximal inhibitory concentrations (IC50s) ofTerminalia chebula extract, Curcuma longa extract, Boswellia serratanon-acidic resin extract (BNRE), Boswellia serrata acidic extractsstandardized to 75-85% total acids and around 5%3-O-acetyl-11-keto-β-boswellic acid (AKBA) and compositions aresummarized in Table 1.

TABLE 1 5-Lipoxygenase enzyme (5-LOX) inhibitory activity S. No. Testedsubstance IC₅₀ (μg/ml) 1 Terminalia chebula extract  26.41 2 Curcumalonga extract  19.53 3 Boswellia serrata non acidic  16.40 resin extract(BNRE) 4 Boswellia serrata extract 5% >100 AKBA 5 Composition-1  14.84 6Composition-2  12.89 7 Composition-3  23.43 8 Composition-4  14.45 9Composition-7  12.35

The above 5-lipoxygenase inhibitory activities in the Table 1, clearlyshow the synergistic efficacy of the compositions comprising Terminaliachebula extract, Curcuma longa extract and Boswellia serrata non-acidicresin extract (BNRE), and those optionally Boswellia serrata acidicextract standardized to 75-85% total acids and/or 5%3-O-acetyl-11-keto-β-boswellic acid (AKBA) in inhibiting the5-lipoxygenase enzyme activity. This observation suggests thatcomposition of the extracts showed greater 5-lipoxygenase inhibitoryactivity than the individual extracts. For example, the IC50 values ofT. chebula extract, C. longa extract, BNRE and Boswellia serrata acidicextract are 25.41 μg/ml, 19.53 μg/ml, 16.40 μg/ml and >100 μg/mlrespectively, whereas, the composition-1, composition-2, composition-3,composition-4 and composition-7 exhibited 5-lipoxygenase inhibitoryactivity with an IC50 value of 14.84 μg/ml, 12.89 μg/ml, 23.43 μg/ml,14.45 μg/ml and 12.35 μg/ml respectively. The 5-lipoxygenase inhibitoryactivity with the compositions is higher than that of the individualextracts.

Example 14: Interleukin 1 (IL-1β) Inhibitory Activity

Inhibition of lipopolysaccharide (LPS)-induced IL-1β production byextracts and compositions was evaluated in Phorbol 12-myristate13-acetate (PMA) differentiated THP-1 human monocytes. Briefly, theTHP-1 cells were differentiated with 20 nM of PMA and incubated furtherfor 48 hrs at 37° C. in a 5% CO2 incubator. Cells were washed with freshDMEM containing 10% fetal calf serum (FCS) and pretreated with differentconcentrations of extracts or compositions. Thereafter, the cells weretreated with Lipo polysaccharides (LPS) (10 ng/ml) and incubated furtherfor 4 hrs. at 37° C. in a 5% CO2 incubator. The supernatant washarvested and the IL-1β concentration secreted into the cell freeculture supernatants was measured using human IL-1β ELISA Kit (R&DSystem, Minneapolis, Minn., USA). Percentage of IL-1β inhibition ofextracts or compositions was calculated from the formula: {(Conc. ofIL-1β in induced—Conc. of IL-1β in the tested extracts orcompositions)×100}÷Conc. of IL-1β in LPS-induced THP-1 human monocytes.The data for inhibition of IL-1β production by extracts or compositionsis depicted in Table 2.

The synergistic efficacy was substantiated by the evaluation of thebiomarker Interleukin-1beta, (IL-1β). The treatment groups supplementedwith composition-1, composition-2, composition-4 and composition-7showed significantly better activity in reducing the serum biomarker,compared to the additive effect contributed by the individualingredients T. chebula extract, C. longa extract, BNRE and Boswelliaserrata acidic extract as shown in Table 2.

TABLE 2 Interleukin 1 (IL-1β) inhibitory activity S. Tested substanceInhibition of IL-1β- No. (50 μg/ml) Production (%) 1 Terminalia chebulaextract  3.36 2 Curcuma longa extract 23.65 3 Boswellia serrata nonacidic  5.16 resin extract (BNRE) 4 Boswellia serrata extract 5% AKBA 4.45 5 Composition-1 22.88 6 Composition-2 19.54 7 Composition-4 20.238 Composition-7 30.20

Referring to Table 2, Composition-1 is administered in a total amount of50 μg/ml, and comprises 50% of the extract of Terminalia chebula (25μg/ml), 25% of the methanol extract of Curcuma longa (12.5 μg/ml), and25% of the Boswellia serrata non-acidic resin extract (BNRE; 12.5μg/ml). If the individual components of Composition-1 had an additiveeffect, 25 μg/ml of Terminalia chebula extract would be expected to be50% as active as 50 μg/ml of pure Terminalia chebula extract. Similarly,12.5 μg/ml of Curcuma longa extract and 12.5 μg/ml of Boswellia serratanon-acidic resin extract would be expected to be 25% as active as 50μg/ml of the pure extracts. Based on this, Composition-1 would beexpected to have a total inhibition of IL-1β production of 8.88%,calculated as follows:

(0.5×3.36% IL-1β (inhibition)+(0.25×23.65% IL-1β inhibition)+(02.5×5.16%IL-1β inhibition)=expected IL-1β inhibition

However, Composition-1 has a total inhibition of IL-1β production of22.88%, which is significantly greater than would be expected from anadditive effect. Thus, Composition-1 shows synergism with regard toinhibition of IL-1β production. Similar calculations demonstrate thatComposition-2, Composition-4, and Composition-7 also show synergism.

This unexpected result clearly indicate that, the synergistic activityof the compositions comprising Terminalia chebula extract, Curcuma longaextract and Boswellia serrata non-acidic resin extract (BNRE) and3-O-acetyl-11-keto-β-boswellic acid (AKBA) in inhibiting the biomarkerInterleukin-1beta (IL-1β) and suggest that compositions are useful forprevention, treatment, inhibition or controlling inflammation and/orimmune related diseases mediated through pro-inflammatorycytokines/chemokines when compare with the individual extracts.

Example 15: Inhibition of Tumor Necrosis Factor-α (TNF-α) In Vitro

The anti-inflammatory activities of extracts and compositions wereassessed in a cell based in vitro assay. Briefly, THP-1 human monocytescells were washed and re-suspended in phenol red free Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 1% fetal Bovine serum(FBS). Equal number of cells was added to each well of a 96-well cellculture plate and the cells were pretreated for 2 h with variousconcentrations of test substances (ranging from 0.5 to 50 μg/ml;solutions prepared in culture medium from a stock solution containing 50mg/1 mL DMSO of each test sample) of extracts or compositions. Theinflammatory response was induced by treatment with 100 ng/ml of LPS for4 h at 37° C. in presence of 5% CO2. The vehicle control culture wellsreceived 0.1% DMSO in culture medium. The cell free culture supernatantswere collected and assessed for secretary pro-inflammatory cytokine,TNFα. The TNF-α concentration was quantitatively measured by highlyspecific and sensitive Enzyme Immuno Assay (EIA) kit supplied by R&DSystems, USA. The enzyme immuno assay was performed based on theprotocol provided by the vendor. The inhibitory concentration for 50%inhibition (IC50) of TNF-α was determined from a plot drawn foringredient concentration against TNF-α level.

Table 3 shows concentrations of compositions for 50% inhibition of TNF-α(IC₅₀) in cell based in vitro model.

TABLE 3 TNFα activity S. Tested substance TNFα activity No. (20 μg/ml)(% of inhibition) 1 Composition-1 51.16 2 Composition-2 72.78 3Composition-3 28.7 4 Composition-4 91.3 5 Composition-5 84.87 6Composition-6 56.01 7 Composition-7 79.55

Example 16: Inhibition of Matrix Metalloproteinase-3 (MMP-3) Activity

Inhibition of MMP-3 production by extracts and compositions wasevaluated in TNFα induced SW982 human synovial cells. Briefly, the SW982cells were cultured in DMEM with 2 mM Glutamine, 100 U/mL penicillin,100 mg/mL streptomycin and 10% fetal bovine serum (Hyclone, Logan,Utah). Five thousand cells per well were seeded into a 96-well cellculture plate (Corning, USA) one day before the experiment. The culturemedia was replaced with fresh DMEM containing 1% fetal bovine serum.Extracts or Compositions were serially diluted in medium, ranging from 5to 100 ng/ml and were pre-incubated with cells for 2 hour at 5% CO2 at37° C., and then stimulated with 10 ng/m1 human recombinant TNFα (R&DSystem, Minneapolis, Minn.) for 24 hours. The cell free culturesupernatants were harvested and used to measure MMP-3 production byELISA development kit (R&D System, Minneapolis, Minn., USA). The MMP-3concentration in cell free culture supernatant was estimatedquantitatively by interpolating the optical densities into the standardcurve generated from known concentrations of MMP-3. The inhibitoryconcentration for 50% inhibition (IC50) of MMP-3 was calculated from theplot constructed by plotting percentage inhibition against concentration(Table 4).

TABLE 4 MMP-3activity S. MMP-3activity No. Tested substance (20 μg/ml)(% of inhibition) 1 Terminalia chebula extract 35.53 2 Curcuma longaextract 44.4 3 Boswellia serrata non acidic 45.22 resin extract (BNRE) 4Boswellia serrata extract 5% 12.98 AKBA 5 Composition-7 66.92

Example 17: The In-Vivo Anti-Inflammatory Activity of Composition-1

The in-vivo anti-inflammatory activity of composition-1, comprising thewater extract of 50 g of Terminalia chebula, 25 g of methanol extract ofCurcuma longa, 25 g of Boswellia serrata non-acidic resin extract(BNRE), so that their percentages in the composition are 50%, 25% and25% respectively was evaluated by an in-vivo study in Freund's CompleteAdjuvant induced arthritis model of Sprague Dawley (SD) rats.

Prednisolone was used as a positive control. The healthy male SD ratswere selected and randomly divided into four groups containing sixanimals per group. The treatment group rats were supplemented daily with200 mg/kg body weight of Composition-1 for 35 days. The positive controlgroup was supplemented daily with Prednisolone at 10 mg/kg body weight.All supplements were diluted in 10 mL of 1% CMC for administration. Theanimals of control group received same volume of 1% CMC. At the 7th day,Freund's Complete Adjuvant (FCA) was injected subcutaneously in thesub-plantar region of the left hind paw of each animal. Paw volumes andweight bearing capacities were respectively measured usingPlethysmography equipment and Incapacitance Meter on the day of FCAinjection and on 35th day of treatment. The difference in volume of pawedema is considered as the inflammatory response. The in-vivoanti-inflammatory response of Composition-1 was estimated by calculatingthe percentage inhibition of paw edema when compared to the CMCsupplemented control

The treatment groups supplemented with 200 mg/kg body weight ofComposition-1 showed 40.35% reduction in paw edema in Table 5.

TABLE 5 S. No Test compound % inhibition of Paw edema 1 Compoisition-140.35% 2 Prednisolone 72.73%

The improvement in percent weight bearing is considered as improvementin efficacy and the results were presented in Table 6.

TABLE 6 S. Percent Weight Bearing No Test compound on Left Hind Limb 1Normal Animals 49.30% 2 Disease control 22.47% 3 Compoisition-1 30.35%

The above in-vivo anti-inflammatory activity values in the Table 5 andTable 6, Supplementation of composition-1 (300 mpk) for 35 days, showeda statistically significant (p<0.05) inhibition of paw edema with 40.35%by the end of day 35 as compared to AIA control.

Supplementation of composition-1 (300 mpk) showed moderate improvementin percentage weight bearing capacity in adjuvant induced arthritisrats.

These results together suggests that oral administration ofcomposition-1 reduces paw edema and improves weight bearing capacity inadjuvant induced arthritis in Sprague Dawley rats.

Example 18: The In-Vivo Anti-Inflammatory Activity of Composition-7

The in-vivo anti-inflammatory activity of composition-7, comprising thewater extract of 36 g of Terminalia chebula, 18 g of methanol extract ofCurcuma longa, 18 g of Boswellia serrata non-acidic resin extract(BNRE), 18 g of Boswellia serrata extract standardized to 75%-85% totalacids by volumetric assay which contain 5%3-O-acetyl-11-keto-β-boswellic acid (AKBA) and 10 g of excipients[Microcrystalline cellulose (MCC) and Syloid], so that their percentagesin the composition are 36%, 18%, 18%, 18% and 10% respectively wasevaluated by an in-vivo study in Freund's Complete Adjuvant inducedarthritis model of Sprague Dawley rats.

Prednisolone was used as a positive control. The healthy male SD ratswere selected and randomly divided into four groups containing sixanimals per group. The treatment group rats were supplemented daily with200 mg/kg body weight of Composition-7 for 35 days. The positive controlgroup was supplemented daily with Prednisolone at 10 mg/kg body weight.All supplements were diluted in 10 mL of 1% CMC for administration. Theanimals of control group received same volume of 1% CMC. At the 7th day,Freund's Complete Adjuvant (FCA) was injected subcutaneously in thesub-plantar region of the left hind paw of each animal. Paw volumes andweight bearing capacities were respectively measured usingPlethysmography equipment and Incapacitance Meter on the day of FCAinjection and on 35th day of treatment. The difference in volume of pawedema is considered as the inflammatory response. The in-vivoanti-inflammatory response of Composition-7 was estimated by calculatingthe percentage inhibition of paw edema when compared to the CMCsupplemented control.

The treatment groups supplemented with 200 mg/kg body weight ofComposition-7 showed 44.34% reduction in paw edema (Table 7).

TABLE 7 S. No Test compound % inhibition of Paw edema 1 Compoisition-744.34% 2 Prednisolone 68.67%

The improvement in percent weight bearing is considered as improvementin efficacy and the results were presented in Table 8.

TABLE 8 S. Percent Weight Bearing No Test compound on Left Hind Limb 1Normal Animals 50.85% 2 Disease control 25.73% 3 Composition-7 45.33%

The above in-vivo anti-inflammatory activity values in the Table 7 andTable 8 indicates, animals administered with composition-7 (200 mpkp.o.) showed 44.34% reduction in paw edema, this reduction in paw edemais statistically significant (p<0.05) as compared to AIA control.Administration of composition-7 (200 mpk p.o.) also resulted 45.33%improvement in weight bearing capacity and this improvement in weightbearing capacity is statistically significant (p<0.05) as compared toAIA control. These results together suggests that oral administration ofcomposition-7 reduces paw edema and improves weight bearing capacity inadjuvant induced arthritis in Sprague Dawley rats.

It will be appreciated to those of ordinary skill in the art thatchanges could be made to the embodiments described above withoutdeparting from the broad inventive concept thereof. It is understood,therefore, that this invention is not limited to the particularembodiments or examples disclosed, but is intended to covermodifications within the objectives and scope of the present invention.

What is claimed is:
 1. A method for treating an inflammation, arthritis,or a joint disease in a patient in need thereof, comprisingadministering to the patient an effective amount of an herbalcomposition, wherein the herbal composition comprises: from 20% byweight to 75% by weight of an extract of Terminalia chebula; from 10% byweight to 40% by weight of an extract of Curcuma longa; and from 10% byweight to 40% by weight of a non-acidic, water-immiscible organicsolvent extract of a Boswellia serrata resin, based on the total weightof the Terminalia chebula, Curcuma longa, and Boswellia serrataextracts.
 2. A method for treating a joint disease in a patient in needthereof, wherein the joint disease is selected from the group consistingof rheumatoid arthritis, psoriatic arthritis, osteoarthritis, refractoryrheumatoid arthritis, chronic non-rheumatoid arthritis,osteoporosis/bone resorption, collagen degradation, gouty arthritis,lupus, juvenile arthritis, joint pain, or a combination thereof, whereinthe method comprises administering to the patient an effective amount ofan herbal composition, wherein the herbal composition comprises: from20% by weight to 75% by weight of an extract of Terminalia chebula; from10% by weight to 40% by weight of an extract of Curcuma longa; and from10% by weight to 40% by weight of a non-acidic, water-immiscible organicsolvent extract of a Boswellia serrata resin, based on the total weightof the Terminalia chebula, Curcuma longa, and Boswellia serrataextracts.
 3. The method according to claim 1, wherein the non-acidic,water-immiscible organic solvent extract of the Boswellia serrata resinis a product obtained by a process comprising: extracting Boswelliaserrata gum resin with a water-immiscible organic solvent to obtain anorganic solution; extracting the organic solution with aqueous alkali toremove boswellic acids from the organic solution; and removing thewater-immiscible organic solvent from the organic solution to obtain anorganic solvent extract.
 4. The method according to claim 1, wherein thenon-acidic, water-immiscible organic solvent extract of the Boswelliaserrata resin is a product obtained by a process comprising: extractingBoswellia serrata gum resin with a water-immiscible organic solvent toobtain an organic solution; extracting the organic solution with aqueousalkali to remove boswellic acids from the organic solution; removing thewater-immiscible organic solvent from the organic solution to obtain anorganic solvent extract; and removing volatile components from theorganic solvent extract under vacuum to obtain said non-acidic,water-immiscible organic solvent extract.
 5. The method according toclaim 1, wherein the herbal composition further comprises at least oneingredient selected from the group consisting of: plant derivedanti-inflammatory agents, pharmaceutically, nutraceutically, ordietetically acceptable carriers, and pharmaceutically, nutraceutically,or dietetically acceptable excipients.
 6. The method according to claim1, wherein: the extract of Terminalia chebula is prepared from driedfruits of Terminalia chebula; the extract of Curcuma longa is preparedfrom dried rhizomes of Curcuma longa; and the non-acidic,water-immiscible organic solvent extract of the Boswellia serrata resinis prepared from a gum resin of Boswellia serrata.
 7. The methodaccording to claim 5, wherein the carriers and excipients are selectedfrom the group consisting of surfactants, binders, diluents,disintegrants, lubricants, preservatives, stabilizers, buffers,suspensions, and drug delivery systems.
 8. The method according to claim1, wherein the herbal composition further comprises: from 0% by weightto 20% by weight of a Boswellia serrata extract which contains at least5% 3-O-acetyl-11-keto-β-boswellic acid (AKBA), based on the total weightof the Terminalia chebula, Curcuma longa, and Boswellia serrataextracts.
 9. The method according to claim 2, wherein the herbalcomposition comprises: from 20% by weight to 75% by weight of theextract of Terminalia chebula; from 15% by weight to 40% by weight ofthe extract of Curcuma longa; and from 10% by weight to 40% by weight ofthe non-acidic, water-immiscible organic solvent extract of theBoswellia serrata resin; and from 0% by weight to 20% by weight of aBoswellia serrata extract which contains at least 5%3-O-acetyl-11-keto-β-boswellic acid (AKBA), based on the total weight ofthe Terminalia chebula, Curcuma longa, and Boswellia serrata extracts.10. The method according to claim 1, wherein the herbal compositioncomprises: from 25% by weight to 65% by weight of the extract ofTerminalia chebula; from 12% by weight to 30% by weight of the extractof Curcuma longa; and from 12% by weight to 30% by weight of thenon-acidic, water-immiscible organic solvent extract of the Boswelliaserrata resin, based on the weight of the composition.
 11. The methodaccording to claim 1, wherein the herbal composition comprises: from 25%by weight to 45% by weight of the extract of Terminalia chebula; from15% by weight to 25% by weight of the extract of Curcuma longa; from 15%by weight to 25% by weight of the non-acidic, water-immiscible organicsolvent extract of the Boswellia serrata resin, and from 15% by weightto 25% by weight of a Boswellia serrata extract which contains at least5% 3-O-acetyl-11-keto-β-boswellic acid (AKBA), based on the weight ofthe composition.
 12. The method according to claim 1, wherein theextracts of Terminalia chebula, Curcuma longa and Boswellia serrata areaqueous, alcoholic or hydroalcoholic extracts.
 13. The method accordingto claim 1, wherein the composition is administered to the patientorally, topically, transdermally, or parenterally.
 14. A method fortreating a patient having an inflammation, arthritis, or a jointdisease, comprising administering to the patient an effective amount ofan herbal composition, wherein the herbal composition comprises: atherapeutically effective amount of an extract of Terminalia chebula; atherapeutically effective amount of an extract of Curcuma longa; and atherapeutically effective amount of a non-acidic, water-immiscibleorganic solvent extract of a Boswellia serrata resin.
 15. A methodaccording to claim 14, wherein the herbal composition is administered toa patient having a joint disease, wherein the joint disease is selectedfrom the group consisting of rheumatoid arthritis, psoriatic arthritis,osteoarthritis, refractory rheumatoid arthritis, chronic non-rheumatoidarthritis, osteoporosis/bone resorption, collagen degradation, goutyarthritis, lupus, juvenile arthritis, joint pain, and a combinationthereof.
 16. The method according to claim 15, wherein the herbalcomposition further comprises from 0% by weight to 20% by weight of aBoswellia serrata extract which contains at least 5%3-O-acetyl-11-keto-β-boswellic acid (AKBA), based on the total weight ofthe Terminalia chebula, Curcuma longa, and Boswellia serrata extracts.